spenito is required for sex determination in Drosophila melanogaster

Abstract

Sex-lethal (Sxl) encodes the master regulator of the sex determination pathway in Drosophila and acts by controlling sex identity in both soma and germ line. In females Sxl maintains its own expression by controlling the alternative splicing of its own mRNA. Here, we identify a novel sex determination gene, spenito (nito) that encodes a SPEN family protein. Loss of nito activity results in stem cell tumors in the female germ line as well as female-to-male somatic transformations. We show that Nito is a ubiquitous nuclear protein that controls the alternative splicing of the Sxl mRNA by interacting with Sxl protein and pre-mRNA, suggesting that it is directly involved in Sxl auto-regulation. Given that SPEN family proteins are frequently mutated in cancers, our results suggest that these factors might be implicated in tumorigenesis through splicing regulation.

Keywords: alternative splicing; germ-line stem cell; sex determination.

Fig. 1.

Nito is essential for ovarian GSC differentiation. (A) Diagram showing the structure of a wild type germarium. (B–B′) WT ovarioles stained for α-Spectrin, Vasa and DAPI. The α-Spectrin antibody labels the round spectrosomes in GSCs (arrow); the Vasa antibody labels all germ cells and DAPI labels nuclei to monitor oocyte (yellow arrowhead) and nurse cell formation (white arrowhead). (C–C′) Egg chambers expressing nito shRNA by MTD-Gal4 were stained for α-Spectrin, Vasa and DAPI. Note the numerous stem-cell-like cells labeled by α-Spectrin and the absence of differentiated nurse cells. (D–E) pH3 staining in WT egg chambers and egg chambers expressing nito shRNA. In WT egg chambers, pH3-positive cells were restricted to the anterior tip of the germarium but were detected throughout nito shRNA egg chambers (arrowheads). (F–F′) Egg chambers expressing Sxl shRNA stained for α-Spectrin, Vasa and DAPI. (Scale bars: 20 μm.)

Fig. S1.

Two independent nito shRNAs result in similar stem-cell-tumor in the germ-line. (A–B′) Egg chambers expressing shRNAs targeting nito (HMJ02081) or nito (HMS02013) using MTD-Gal4 stained for α-Spectrin, Vasa and DAPI. (Scale bars: 20 μm.)

Fig. 2.

Nito is required for sex determination in somatic tissues. (A and B) dome-Gal4 drives expression in the first pair of leg discs (A) and genital discs (B), as shown by UAS-2xEYFP. (C) Foreleg of a WT male with the dark thickened sex comb bristles. (D) Foreleg of a WT female. Note the absence of sex combs. (E) Foreleg of a female fly expressing nito shRNA driven by dome-Gal4. Some bristles are transformed into male sex combs. (F and G) Genitalia of wild-type male (F) and female (G) flies showing distinct morphology, such as penis apparatus and claspers in male (F, arrow and arrowhead, respectively) and vaginal bristles in female (G, arrow). (H) nito shRNA driven by dome-Gal4 transforms female genital morphology into male-like, as evidenced by the absence of vaginal bristles and appearance of structures resembling penis apparatus (arrow) and claspers (arrowhead).

Fig. 3.

Nito is a ubiquitous nuclear protein that shows no sex-biased expression. (A) Schematic diagram showing domain structures of Spen and Nito proteins and the peptide used to generate the Nito antibody. (B) nito mRNA levels in male and female WT wing discs were measured by qRT-PCR. Error bars represent SDs. (C) Nito antibody recognizes a single band of the predicted ∼89 kDa size in S2 cell lysates. (D–F) Nito antibody stainings in WT female (D) or male (E) wing discs and ovarioles (F). (G–G′) Expression of Sxl shRNA in the dorsal half of the wing disk (below the dashed line) using ap-Gal4 leads to depletion of Sxl protein, but has no effect on Nito protein levels. (H–H′) Nito antibody staining in wing discs containing nito¹ mutant clones, marked by the absence of GFP. Note the absence of Nito staining in nito¹ clones.

Fig. 4.

Nito is required for Sxl levels and regulates Sxl mRNA splicing. (A and B) Sxl stainings in WT male (A) and female (B) wing discs. (C–C′) Expressing nito shRNA in the dorsal half of the disk (below the dashed line) using ap-Gal4 leads to strong reduction of Nito (C) and Sxl (C′) stainings. (D–D′) Sxl antibody staining (D′) in wing discs containing nito¹ mutant clones, which are marked by the absence of GFP (D). Note the absence of Nito and Sxl staining in nito¹ clones. (E and F) Sxl stainings in WT egg chambers (E) or in egg chambers expressing nito shRNA by MTD-Gal4 (F). Scale bars: 20 μm. (G) Diagram showing the alternative splicing event that produces the male- or female-specific Sxl transcripts. The arrows indicate the primers used for RT-PCR. Sxl splicing was analyzed by RT-PCR using RNA extracted from wing discs or ovaries. Male-specific bands: 2–3-4. Female-specific bands: 2–4.

Fig. S2.

nito, but not spen, regulates Sxl levels in wing discs. (A–B′) Expression of nito shRNA (HMJ02081 or HMS02013) in the dorsal half of the wing disk using ap-Gal4 leads to a strong reduction of both Nito (A and B) and Sxl (A′ and B′) stainings. (C) Expression of spen shRNA in the dorsal half of the disk (below the dashed line) by ap-Gal4 does not affect Sxl protein levels. spen shRNA generates embryonic lethality with cuticle and head defects when expressed using MTD-Gal4 (47), which resembles the phenotype of the spen mutant, indicating that the shRNA is functional.

Fig. 5.

Nito interacts with Sxl and Sxl pre-mRNA in S2 cells. (A) HA-Sxl, GFP-Nito or GFP expression vectors were transfected individually or together into Drosophila S2 cells. Cell lysates were immunoprecipitated using GFP nanobody or anti-HA antibody and analyzed by Western blot. GFP alone is used as a control. Asterisk indicates IgG heavy chain. (B) S2 cells were transfected with GFP-Nito and HA-Sxl, and Co-IP was performed using GFP nanobody in the absence or presence of RNase A and RNase T1. (C) S2 cells were transfected with GFP-Nito, GFP-Sxl or GFP and immunoprecipitated with GFP nanobody. The presence of Sxl pre-mRNA was detected by RT-PCR using an intron 3/exon 4 primer pair. GFP-Sxl was used as a positive control and GFP alone as a negative control. (D) Model: Nito forms a complex with Sxl and together they repress the splicing of Sxl exon 3 in female tissues.

Fig. S3.

Nito shRNA affects wing growth more strongly in females than in males. (A) WT male wing. (B) WT female wing. (C) Overlay of the images in A (red) and B (blue) shows that a WT female wing is about 30% larger than a WT male wing. Male (D) and female (E) wings in which nito shRNA was expressed using the nub-Gal4 driver. (F) Overlay of D and E showing that both male and female wings reach about the same size upon nito knockdown.